Proteins are universally dynamic structures, and their conformational fluctuations play central roles in a wide array of protein functions, both enzymatic and non-enzymatic. Receptors and signaling proteins, in particular, are designed to have flexible, dynamic structures which can be allosterically switched between 'on' and 'off' states. An understanding of the conformational changes and thermal dynamics of these proteins is essential to a description of their function in both diseased and healthy cellular systems. Cellular transformation in cancer, for example, often results from a receptor or signaling protein which is locked into an 'on' state by a specific mutation, thereby preventing the normal conformational change to the 'off' state. Such activating mutations are known in prokaryotic signaling systems, and are a key focus of the present proposal. The primary goal of the proposed research is to provide general physical insights into the conformational dynamics and activation of receptors and signaling proteins. The E. coli chemosensory pathway provides a set of proteins characterized by X-ray structures, but with unknown dynamics and activation mechanisms. These proteins are well-suited for an approach which uses engineered disulfide bonds to trap long-range backbone motions, to map out contacts between transmembrane helices, and to lock signaling states. Further, quite complementary information is provided by NMR techniques, which are used to probe the effects of activation on conformation and dynamics. The specific aims apply these techniques to three chemosensory proteins: the galactose binding protein, the transmembrane aspartate receptor, and the phospho-signaling protein CheY. Conformational changes being examined include those triggered by ligand binding and phosphorylation, as well as by specific mutations which constitutively produce the 'on' state. Parallel studies are investigating thermal backbone motions in regions of each protein critical to activation. These proteins are of interest in part because they are closely related to chemosensory proteins in pathogenic bacteria, which are potential targets for antimicrobial agents designed to disrupt chemotaxis. In addition, the E. coli components exhibit interesting parallels with certain eukaryotic receptors and signaling proteins, such as the insulin receptor and H-ras, respectively.